Gliederung

Objective

The mitogenic MAPK-cascade plays a major role in regulation of proliferation, differentiation and apoptosis. Recently, the MAPK Erk has been shown to be directly involved in regulation of genes for migration relevant proteins. Since mutated B-Raf proteins are involved in development of melanomas and colon carcinomas, we were interested in alterations of the expression of specific signalling proteins and their function in astrocytic tumors.

Methods

For detection of alterations at gene-expression level 10 astrocytomas grade II and 10 glioblastoma multiforme were screened by RT-PCR. The function of detected proteins was explored by performing overexpression and RNAi knock-down studies in cell culture. We used electroporation by nucleofector technology (Amaxa) for high efficiency transfection of U251 glioblastoma cells with pCMV5-expression plasmids, and we designed and validated siRNAs for gene silencing. We then explored the influence of detected proteins on cell-proliferation and migration in cell based assays by overexpression and silencing.

Results

We discovered that A-Raf is highly overexpressed in 60% of analysed astrocytomas and 33% of glioblastomas, whereas only very low expression of A-Raf was found in normal brain tissue. Our experiments did not reveal any differences in proliferation or migration between A-Raf overexpressing or A-Raf deficient cells compared to control cells. However, there are hints that A-Raf may play a role in regulation of the tumor-cell's metabolism. As our previous experiments showed, A-Raf interacts directly with pyruvate kinase M2, a tumor-specific metabolic enzyme, which is consistent with data published about the mitochondrial localization of A-Raf. In addition, A-Raf is part of the glycolytic complex.

Conclusions

The overexpression of A-Raf in tumor specimens of astrocytoma grade II and glioblastoma multiforme clearly shows that this protein kinase plays a role in the development of brain tumors. However, its function remains obscure. Our data suggest that A-Raf is not involved in regulation of migration or proliferation of the cancer cell and therefore must have a different function. There are indications that A-Raf may be involved in regulation of the tumor cell's metabolism. A change in the metabolic rate of glial cells may be a factor triggering tumor formation.